The site-specific DNA restriction endonuclease, endoR.HhaII, from the bacterium Haemophilus haemolyticus is to be used to determine the chemical basis of how protein molecules recognize specific sequences in DNA. The gene for the enzyme along with the gene for its corresponding DNA modification methylase has been cloned into a plasmid vector using recombinant DNA techniques. With the restriction endonuclease gene cloned on a plasmid, it is possible to mutagenize the recombinant plasmid in vitro, introduce the mutagenized plasmid into an appropriate bacterial host and select for conditionally lethal (temperature-sensitive) mutants. Among these should be a class of mutants in which the restriction endonuclease has suffered an alteration in its ability to recognize the proper site in DNA (but only when the temperature is raised). The enzyme would cleave the DNA of the host with broadened specificity resulting in cell death. A collection of such mutant clones would be analyzed to locate the nature and position of all of the mutations in the primary structure of the restriction enzyme protein. What would emerge would be an amino acid map of sites in the protein which are responsible for mediating the sequence recognizing ability of the enzyme. These data would be used in conjunction with the results of three-dimensional X-ray diffraction studies performed on crystals of endoR.HhaII protein. The two approaches together should elucidate the nature of the chemical contacts made by the enzyme with its DNA substrate. This information should be useful in understanding how other sequence-specific proteins interact with DNA, especially those controlling the action of genes.